Occurrence of Neofusicoccum parvum Causing Canker and Branch Dieback of European Hazelnut in Maule Region, Chile.

The European hazelnut (Corylus avellana) is an important fruit crop cultivated in Chile, with over 17,000 ha planted (46%) in the Maule region, central Chile. During a routine orchard survey in seasons 2020-2021 and 2021-2022, in the Maule region, canker and dieback symptoms were observed in two commercial orchards of European hazelnut cv. Tonda Di Giffoni in San Rafael (8-year-olds) and Linares (15-year-olds), with an incidence between 10% and 36%, respectively, based on external symptoms. Twenty symptomatic branches exhibiting cankers, reduced vigor, wilting, twig death, and dieback, were collected. A cross-section of diseased branches revealed mostly brown V or U-shaped cankers of hard consistency. Branches were cut, and pieces of cankers were surface sterilized in 96% ethanol for 3 s and briefly flamed. Small pieces of wood (5 mm2) from the edge of cankered tissues were placed on Potato Dextrose Agar (2% PDA) amended with 0.1% Igepal CO-630 and incubated at 25°C for five days in the dark (Díaz and Latorre 2014). Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Eight pure cultures (NP-Haz01 to NP-Haz08) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA under near-UV light. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown globose pycnidia after 21 days at 25°C. Conidia were hyaline, aseptate, ellipsoidal, densely granulate, externally smooth, and thin-walled dark, that measured (9.5-) 15.5 ±1.2 (-17.3) x (5.1-) 7.2 ± 0.6 (-9.1) µm (n = 30), with a length/width ratio of 2.15. These isolates were tentatively identified morphologically as Neofusicoccum sp. Molecular identification was performed using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R primers of the internal transcribed spacer (ITS1-5.8S-ITS2) region, a portion of the beta-tubulin (BT) and part of the translation elongation factor (EF1-) genes, respectively (Dissanayake et al. 2015). A MegaBlast search in GenBank showed a 99% similarity to isolate CMW9081, the ex-type of Neofusicocum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. The sequences were added to GenBank (OR393855 to OR393857 for ITS; OR400688 to OR400690 for BT; OR400691 to OR400693 for EF1-). Pathogenicity of three isolates (NP-Haz02, NP-Haz04, NP-Haz09) was studied on freshly made pruning wounds on attached branches of 3-year-old and one-year-old of European hazelnut cv. Tonda Di Giffoni in the San Rafael field. Fifteen pruning wounds were inoculated with 40 µL conidial suspension (105 conidia/mL) of each isolate of N. parvum. Sterile distilled water was used as a control treatment (n=15 branches) for branches of 3-year-olds and one-year-olds. Both pathogenicity tests were repeated once. Attached branches of 3-year-olds (6 months of incubation) and one-year-olds (4 months of incubation), developed necrotic streaks and cankers with a mean length of 33 to 82 mm and 25 to 51 mm, respectively. No necrotic streaks were observed in the branches treated with water. Neofusicoccum parvum was reisolated only from symptomatic tissues of inoculated branches, and morphological and molecularly (EF1-) identified, thus fulfilling Koch's postulates. Previously, other Botryosphaeriaceae spp. as Diplodia coryli (Guerrero and Pérez 2012) and D. mutila (Moya-Elizondo et al. 2023) have been obtained from canker and dieback of hazelnut in Chile. Recently, N. parvum was reported causing nut rot in hazelnuts in Italy (Wagas et al. 2022). To our knowledge, this is the first report of N. parvum causing canker and branch dieback of hazelnut trees in Chile and worldwide.

Occurrence of Alternaria alternata and A. tenuissima causing black rot in cherry fruits (Prunus avium) in Central Chile.

During the harvest of 2020 and 2021, sweet cherry (Prunus avium) fruit showed a firm rot with irregular pale to dark brown lesions on the fruit surface, with green to light brown fungal growth resembling Alternaria-like infection (Simmons, 2007). Diseased cherries (n= 80 fruit) were collected at harvest in mature (over 10-year-old) commercial orchards of cherry tree varieties Lapins, Regina, Santina, Skeena, and Sweetheart planted in four localities of the regions O´Higgins (33°59´ S, 70°42´W; San Francisco de Mostazal and Graneros) and Maule (35°00'S, 71°23´W; Curicó and Sagrada Familia), Central Chile. The incidence of black rot was 1.9 and 3.2% in O´Higgins and Maule region, respectively, and it was increased to up to 5% during cold storage. The fruit collected previously, were transported to the lab, and surface disinfected in 75% ethanol for 15 s, and rinsed in sterile water. Internal pieces from the junction of diseased and healthy tissues of fruits were placed on potato dextrose agar (PDA, 2%) for 7 days at 20°C. Forty-two isolates of Alternaria-like (Simmons, 2007) were recovered consistently from pure cultures taking hyphal tips from 7 days old cultures. On PDA, 28 isolates (group A) were characterized by cottony, white-gray to green colonies and conidial chains (4 to 10 conidia) with secondary chains (1 to 5 conidia) branching on the conidiophore. Conidia were ovate to obclavate (mean 22.8 ± 5.1 x 8.8 ± 1.5 µm; n=40) with 3 to 7 transepta and 1 longisepta. The remaining 14 isolates (group B) were characterized by cottony, olive-green to olive-brown colonies following a ring pattern of growth and white margins, with conidial chains (4 to 14 conidia) and uncommon secondary chains (1 to 4 conidia). Conidia were obpyriform to ovate, light brown to brown with a cylindrical short beak at the tip (mean 24.7 ± 5.9 × 11.2 ± 1.3 µm; n=40) with 2 to 4 transepta, and 0 to 2 longisepta. Two representative isolates of group A (Sant-02-2020 and Bing-03-2020) and group B (Sant-26-2021 and Skeen-43-2021) were amplified for the Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), and calmodulin (Cal) loci following the protocols described by Hong et al. (2005) and Lawrence et al. (2013). A MegaBlast search of sequences of group A (GenBank nos. OR267293- OR267294, OR258001- OR258002, and OR267297- OR267298, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains UCD10529 and UCD10539 of A. alternata, and group B (GenBank nos. OR267295- OR267296, OR258003- OR258004, and OR258005- OR258006, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains EGS 34-015 and A30 of A. tenuissima. Combined phylogenetic analysis using MEGA X clustered isolates Sant-02-2020 and Bing-03-2020, and Sant-26-2021 and Skeen-43-2021 with ex-type of A. alternata and A. tenuissima, respectively. Pathogenicity tests were conducted using isolates of A. alternata (Sant-02-2020; Bing-03-2020) and A. tenuissima (Sant-26-2021; Skeen-43-2021). Detached ripe cherry fruit var. Sweetheart (n=40 fruits/isolate) and Regina (n=40 fruits/isolate) were surfaces disinfested (75% ethanol, 30 s), wounded in the middle with a sterile needle (2 mm in depth), and inoculated with 20 µL of conidial suspension (106 conidia/mL). An equal number of healthy cherries (n=40 fruits) treated with sterile water were used as controls. The experiment was repeated once. All inoculated fruit incubated for 7 days at 22°C, developed between 13 ± 2.7 to 23 ± 2.5 mm and 14.1 ± 1.1 to 19 ± 3.6 mm in lesion diameter for A. alternata and A. tenuissima isolates, respectively. Koch´s postulates were fulfilled by 100% reisolation of the causal pathogen from inoculated fruit, and molecular identification of A. alternata and A. tenuissima isolates. Previously, A. alternata has been described as causing rots on cherries in Chile (Acuña 2010), and China (Zhao and Liu, 2012; Ahmad et al., 2020). To our knowledge, this is the first occurrence of cherry black rot caused by A. alternata and A. tenuissima in Central Chile. Epidemiological studies are necessary to develop integrated management of cherry black rot in Central Chile.

Dothiorella sarmentorum Causing Branch Dieback of English Walnut in Maule Region, Chile.

English walnut (Juglans regia), cv. Chandler is the most cultivated tree nut in Chile, with 43,734 ha. In Maule Region, central Chile, English walnut plantings have expanded over an additional 7,000 ha in the last five years. During a routine orchard survey in 2019, branch and twig dieback symptoms were observed in two commercial orchards located in San Rafael (10 years old) and Longaví (12 years old) in the Maule Region, with an incidence of 45% to 65% of affected trees, respectively. Symptomatic branch samples (n = 15) were collected from the two commercial orchards and transported to the laboratory in a cooler and then surface sterilized in 96% ethanol for 3 s and briefly flamed. Cross-section of symptomatic branches revealed brown to dark-brown wedge-shaped wood cankers. Small (5 mm) pieces of wood from the edge of cankered tissues were placed on Potato Dextrose Agar (PDA, 2%) amended with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (PDAm) (Díaz and Latorre 2014) and incubated at 25°C for five days in the dark. Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Each fungal isolate was recovered from a single diseased branch (47%). Seven isolates (Dsar-1 to Dsar-7) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown pycnidia after 15 days at 25°C. Conidia were hyaline and aseptate, dark brown, 1-septate, with a brown wall, ovoid with a broadly rounded apex and truncated base, (17.5-) 19.5 ±1.2 (-22.0) x (7.6-) 8.9 ± 0.6 (-10.1) µm (n = 30). These isolates were tentatively identified morphologically as Dothiorella sp. (Phillips et al. 2005). Molecular identification was performed using ITS1/ITS4 and EF1-728F/EF1-986R primers (White et al. 1990; Dissanayake et al. 2015) of the internal transcribed spacer (ITS1-5.8S-ITS2) region and part of the translation elongation factor (EF1-) genes, respectively. A MegaBlast search in GenBank showed a 100% similarity to isolate CBS 115038, the ex-type of Dothiorella sarmentorum. The sequences were added to GenBank (OM161950 to OM161956 for ITS; OM177188 to OM177194 for EF1-). Pathogenicity of two isolates (Dsar-2 and Dsar-7) was tested in the orchard on freshly made pruning wounds on attached branches of 2-year-old-pruned English walnut trees cv. Chandler. A second pathogenicity test was done on freshly made pruning wounds in 1-year-old rooted cuttings (n=15) (40 cm of long) of English walnut cv. Chandler. Each pruning wound was inoculated with 40 µL conidial suspension (105 conidia/mL). Sterile distilled water was used as a control treatment. Both pathogenicity tests were repeated once. After seven months for attached branches and four months for rooted plants, necrotic streaks with a mean length of 81.3 and 44.5 mm were observed below the inoculated pruning wounds, respectively. No necrotic streaks were observed in any of the control wounds. Dothiorella sarmentorum was 100% reisolated from symptomatic tissues of inoculated branches and molecularly identified (EF1-), thus fulfilling Koch's postulates. Recently, D. sarmentorum has been reported causing English walnut dieback in Spain (López-Moral et al. 2020). To our knowledge, this is the first report of D. sarmentorum causing branch dieback of English walnut in Chile. Further studies are needed to know the impact and extent of canker and branch dieback of walnut in commercial orchards in the Maule Region, central Chile.

Severe Outbreak of Dry Core Rot in Apple Fruits cv. Fuji Caused by Kalmusia variispora During Pre-harvest in Maule Region, Chile.

Apple (Malus × domestica) is an important fruit crop in Chile, with a cultivated area of 32,313 ha concentrated (63%) in Maule Region (35°25' S). Unusual core rot on 'Fuji' apples was observed at harvest in a commercial orchard in Curicó, Maule Region, with an incidence ranged between 22 to 35% in 2018 and 2019. Previously, in 2017, an incidence of 30% was estimated on 'Fuji' fruits destined to the Asian market. Internal decay symptoms consisted of dry, corky light to dark-brown tissue, within the seed locules initially. In moderate to severe cases, the necrotic lesion progresses deeper into the mesoderm. External symptoms were quite subtle, and typically, the disease goes unnoticed. However, infected fruit ripen earlier. Small pieces (2-3 mm) from the internal lesion margin of symptomatic apples (n = 50) were placed on Potato Dextrose Agar (PDA) (2%) and incubated at 20°C for 10 days. Pure cultures (n = 41) were obtained and transferred to Malt Extract Agar (MEA) (2%). Colonies on MEA produced an even to slight undulating buff margin with white woolly aerial mycelium, and immerse ochreous in the center, changing gray to olivaceous aerial mycelium with age. On the underside, colonies were umber and buff in the center and margin, respectively. After 10 days, numerous densely aggregate dark-brown mature pycnidia were observed. Aseptate conidia were subglobose to cylindrical, straight, and sometimes curved with rounded at both ends, that was initially hyaline to pale olive, thin, smooth wall with mean dimensions of (2.9-) 3.4 (-4.4) x (1.5-) 1.8 (2.2) µm (n=50). Based on morphology, the fungus was identified as Kalmusia variispora (Verkley et al. 2014). The internal transcribed space (ITS), portion of ß-tubulin (TUB), and large subunits of the nuclear ribosomal RNA (LSU) loci were used for molecular identification, using primers ITS4/ITS5, Bt2a/Bt2b, and LR0R/LR5 (Ariyawansa et al., 2014). BLAST searches indicated 100% identity with K. variispora (ex-type CBS 121517). The maximum parsimony phylogenetic analysis placed Chilean isolates in the K. variispora clade. Sequences were deposited in GenBank (OL711706 to OL711709, OL739499 to OL739502 and OL711710 to OL711713 for ITS, TUB and LSU, respectively). Pathogenicity tests were conducted using four K. variispora isolates. 'Fuji' apples (n = 20) were surface disinfested (75% ethanol, 30 s) and then wounded and inoculated with conidial suspension (50 L of 106 conidia/mL) deposited in the middle and into the core region using a sterile fine-tipped micropipette. Additionally, 20 one-year dormant rooted cuttings 'Fuji' and 'Cripps Pink' were pruned and immediately inoculated on the pruning wound. An equal number of apples and rooted cuttings treated with sterile water were used as controls. The experiments were repeated once. All inoculated fruits developed lateral lesions (22 to 37 mm) and dry core rot (18 to 36 mm) symptoms identical to those described in the original outbreak, after 20 days at 20°C in a commercial packing box. The inoculated cuttings produced canker lesions of 10 to 21 mm in length, and dieback symptoms were observed after 3 months. No symptoms were observed on the negative controls. Koch's postulates were fulfilled by 100% reisolating K. variispora. Previously, Alternaria spp. have been reported as the primary pathogen associated with moldy core and dry core rot of apples worldwide (McLeod et al., 2014) and in Chile (Elfar et al., 2018). However, Kalmusia spp. have been associated with dry core rot in apples (McLeod et al., 2014) and have been isolated from canker symptoms on apples in Chile (Díaz et al. 2021). To our knowledge, this is the first report of a severe outbreak of K. variispora causing dry core rot in apples in Chile and worldwide.

Characterization and Pathogenicity of Diplodia, Lasiodiplodia, and Neofusicoccum Species Causing Botryosphaeria Canker and Dieback of Apple Trees in Central Chile.

In recent years, the number of apple trees affected by Botryosphaeria cankers and dieback has considerably increased in central Chile. This study aimed to identify the species of Botryosphaeriaceae associated with canker and dieback symptoms, estimate disease incidence and distributions, and study their pathogenicity and virulence on apple and other fruit crops. A field survey of 34 commercial orchards of apple (7 to 30 years old) was conducted in 16 localities, obtaining 270 symptomatic branch and trunk samples in 2017 and 2018 growing seasons. The incidence of Botryosphaeria canker and dieback ranged between 5 and 40%, and a total of 255 isolates of Botryosphaeriaceae spp. were obtained from 238 cankers. Morphological identification along with phylogenetic studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the translation elongation factor 1-α (tef1-α), and part of the ß-tubulin (tub2) genes allowed us to identify Diplodia mutila (n = 49 isolates), D. seriata (n = 136 isolates), Lasiodiplodia theobromae (n = 16 isolates), and Neofusicoccum arbuti (n = 54 isolates). L. theobromae was isolated mainly from apple dieback from northern localities. All pathogens tested were pathogenic, causing canker and dieback symptoms on lignified twigs of apple, pear, walnut, and green grapevine shoots in the field. Isolates of N. arbuti were the most virulent, reproducing more severe cankers on the lignified tissues inoculated. This study reports, for the first time, D. mutila and L. theobromae associated with Botryosphaeria canker and dieback in Chile, and it is the first description of N. arbuti causing apple dieback worldwide.

Severe outbreak of Fusarium wilt on common beans (Phaseolus vulgaris) caused by Fusarium oxysporum in the Maule Region, central Chile.

Common bean (Phaseolus vulgaris L.) is an important grain legume cultivated worldwide as food for humans and livestock (Schwartz et al., 2005). Common beans in central Chile reach up to 3,893 ha from which 1,069 ha are located in the Maule region. Common bean is produced by small farmers who have limited access to fertilization, technical irrigation, and crop protection. In spring 2018, bean plants initially showed a slight yellowing and premature senescence 50 days after sowing (das) until showing wilting symptoms (70 -100 das) in Curepto fields (35 05'S; 72 01'W), Maule region. The basal part of affected plants displayed internal reddish-brown discoloration of the vascular tissues. Based on the plant external symptoms, we estimated an incidence between 15% and 45% in bean fields. Nine symptomatic plants were collected, and surface washed with sterile water and disinfested with 75% ethanol (v/v). Then small fragments (5-mm) from damage vascular tissue from each plant were cut and placed on Petri dishes containing PDA acidified with 0.5 ml/l of 92% lactic acid (APDA, 2%). The isolations were incubated for seven days at 25°C. Nine Fusarium-like isolates from single-spore on APDA (2%) became pale vinaceous, floccose with abundant aerial mycelium and dark vinaceous reverse colony, with a growing rate of 10.8 to 11.6 mm/d at 25°C (Lombard et al., 2019). Phialides were short, singular growing laterally on the mycelium. Macroconidia were hyaline, fusiform with basal foot cells shaped to pointed and apical cells tapered, 2-5 septate, and 28.6 to 47.6 (av. 38.1) µm long x 2.2 to 3.6 (av. 3.1) µm wide. Microconidia were hyaline, oval to ellipsoid, one-celled, and 4.5 to 10.9 (av. 6.1) µm long and 2.2 to 3.3 (av. 2.7) µm wide (n=50 spore). For molecular identification, three isolates (Curi-3.1, Be-8.1, and Be-11.3) were sequenced using PCR amplification of the partial sequences of beta-tubulin (BT) and translation elongation factor 1-α gene (TEF) (Lombard et al., 2019). NCBI BLAST analysis showed 99 to 100% similarity with sequences (TEF; BT) of strain CPC 25822 of Fusarium oxysporum. The maximum-likelihood phylogenetic analysis placed the Chilean isolates in the F. oxysporum complex clade. Chilean sequences were deposited into GenBank under accession numbers MW419125, MW419126, MW419127 (TEF) and MW419128, MW419129, MW419130 (BT). Pathogenicity tests (isolates Curi-3.1, Be-8.1, and Be-11.3) were conducted under greenhouse (15-28°C, 85%RH) on healthy bean plants (n=30) cv. Blanco Español INIA cultivated in pots (sand/peat moss/soil) at the University of Talca. Plants that are 30 days-old were inoculated using 200 µl of conidial suspension (106 conidia/ml) on wounded roots (crown). Control plants (n=10) were similarly inoculated with sterile distilled water. After 45 days, all inoculated plants with F. oxysporum isolates developed necrotic lesions on vascular tissue, and chlorosis, and wilting while control plants remained healthy. This experiment was conducted twice. The pathogen was reisolated (100%) from diseased plants and molecularly identified as F. oxysporum. To our knowledge, this is the report of a severe outbreak of F. oxysporum causing Fusarium yellows in P. vulgaris in the Maule region, Chile. Previously, F. oxysporum has been reported affecting tomato (Sepúlveda-Chavera et al., 2014) and blueberry in Chile (Moya-Elizondo et al., 2019).

Identification and Pathogenicity of Diplodia, Neofusicoccum, Cadophora, and Diaporthe Species Associated with Cordon Dieback in Kiwifruit cultivar Hayward in Central Chile.

Dieback symptoms associated with fungal trunk pathogens cause significant economic losses for farmers of kiwifruit and other woody fruit trees worldwide. This study represents the first attempt to identify and characterize the fungal trunk pathogens associated with cordon dieback disease of kiwifruit in central Chile. Field surveys were conducted throughout the main kiwifruit-growing regions in central Chile to determine the incidence and characterize the fungal trunk pathogens associated with cordon dieback of kiwifruit cultivar Hayward through morphological, molecular, and pathogenicity studies. A total of 250 cordon samples were collected and isolations were performed on 2% acidified potato dextrose agar (APDA) plus antibiotics and Igepal. The incidence of kiwifruit cordon dieback ranged between 5% and 85% in all surveyed areas in central Chile. A total of 246 isolates were isolated and identified using culture and morphological features belonging to three fungal taxa: Diaporthaceae spp. (Diaporthe ambigua and D. australafricana; n = 133 isolates); Botryosphaeriaceae spp. (Diplodia seriata and Neofusicoccum parvum; n = 89 isolates); and Ploettnerulaceae spp. (Cadophora luteo-olivacea and C. malorum; n = 24 isolates). These were identified using phylogenetics studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the ß-tubulin gene (tub2), and part of the translation elongation factor 1-α gene (tef1-α). Isolates of N. parvum and D. seriata were the most virulent, causing internal brown lesions and dieback symptoms in attached green shoots, attached lignified canes, and young inoculated kiwifruits. This report is the first to describe D. seriata and C. luteo-olivacea associated with kiwifruit cordon dieback in Chile. It presents the first description of N. parvum causing kiwifruit dieback worldwide.

Priming of Defense Systems and Upregulation of MYC2 and JAZ1 Genes after Botrytis cinerea Inoculation in Methyl Jasmonate-Treated Strawberry Fruits.

Several attempts have been made to study the effects of methyl jasmonate (MeJA) on plants in the past years. However, the comparative effects of the number and phenological time of MeJA applications on the activation of defense systems is currently unknown in strawberries. In the present research, we performed three field treatments during strawberry (Fragaria× ananassa 'Camarosa') fruit development and ripening which consisted of differential MeJA applications at flowering (M3), and the large green (M2 and M3) and red ripe (M1, M2, and M3) fruit stages. We also checked changes in gene expression related to plant defense against Botrytis cinerea inoculation post-harvest. In M3 treatment, we observed an upregulation of the anthocyanin and lignin contents and the defense-related genes, encoding for chitinases, ß-1,3-glucanases and polygalacturonase-inhibiting proteins, after harvest (0 hpi), along with the jasmonate signaling-related genes FaMYC2 and FaJAZ1 at 48 h after B. cinerea inoculation (48 hpi) during postharvest storage. Although we did not find differences in gray mold incidence between the MeJA treatments and control, these results suggest that preharvest MeJA treatment from the flowering stage onwards (M3) primes defense responses mediated by the upregulation of different defense-related genes and retains the upregulation of MYC2 and JAZ1 at 48 hpi.

Biocontrol Potential of Grapevine Endophytic and Rhizospheric Fungi Against Trunk Pathogens.

Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

Identification of Virulences of the Rust Fungus Melampsora larici-populina Occurring in Chile.

Melampsora larici-populina Kleb. is a Eurasian species that causes leaf rust of many species of Populus from sections Tacamahaca and Aigeiros. This rust has been inadvertently introduced to many parts around the world affecting poplar species that grow naturally, which may be susceptible to this fungus. In Chile, early European settlers introduced poplars; rust, attributed to M. larici-populina, has been reported in these trees since 1918. However, a modern confirmation has been lacking, and pathogenic variation of the Chilean population of M. larici-populina has not been investigated. Using a morphological analysis of urediniospores and sequencing of internal transcribed spacer (ITS) regions, we confirmed the presence of M. larici-populina in Chile. ITS regions exhibited 100% homology with M. larici-populina. Scanning electron microscopy showed that spores were 26 to 47 µm in length, 13 to 16 µm in width, and echinulate except for apices, which are smooth, characteristics described for this species of rust. The variability of M. larici-populina is characterized by the presence of pathotypes, which allows the fungus to infect despite the resistance of certain poplar hybrids. We concluded that the identified spores belong to M. larici-populina, with virulences 1, 2, 3, 4, 5, and 6. These results describe variation in virulence of M. larici-populina, which suggests the presence of a sexual stage in Chile.

Evaluation of a decision support strategy for the control of powdery mildew, Erysiphe necator (Schw.) Burr., in grapevine in the central region of Chile.

BACKGROUND: The primary strategy to control powdery mildew in Chilean vineyards involves periodic fungicide spraying, which may lead to many environmental and human health risks. This study aimed to implement and evaluate the effectiveness and economic feasibility of a novel decision support strategy (DSS) to limit the number of treatments against this pathogen. An experiment was conducted between the 2010 and 2013 seasons in two irrigated vine fields, one containing a cultivar of Cabernet Sauvignon (CS) and the other a cultivar of Chardonnay (CH). RESULTS: The results showed that the DSS effectively controlled powdery mildew in CS and CH vine fields, as evidenced by a disease severity lower than 3%, which was lower than that observed in untreated vines (approximately 10 and 40% for CS and CH respectively). The DS strategy required the application of only 2-3 fungicide treatments per season in key vine phenological stages, and the cost fluctuated between $US 322 and 415 ha-1 , which was 40-60% cheaper than the traditional strategy employed by vine growers. CONCLUSION: The decision support strategy evaluated in this trial allows a good control of powdery mildew for various types of epidemic with an early and late initiation. © 2017 Society of Chemical Industry.

Identification and Characterization of Diaporthe ambigua, D. australafricana, D. novem, and D. rudis Causing a Postharvest Fruit Rot in Kiwifruit.

Diaporthe spp. are important plant pathogens causing wood cankers, blight, dieback, and fruit rot in a wide range of hosts. During surveys conducted during the 2013 and 2014 seasons, a postharvest rot in Hayward kiwifruit (Actinidia deliciosa) was observed in Chile. In order to identify the species of Diaporthe associated with this fruit rot, symptomatic fruit were collected from seven kiwifruit packinghouses located between San Francisco de Mostazal and Curicó (central Chile). Twenty-four isolates of Diaporthe spp. were identified from infected fruit based on morphological and cultural characters and analyses of nucleotides sequences of three loci, including the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequences of the ß-tubulin, and translation elongation factor 1-α genes. The Diaporthe spp. identified were Diaporthe ambigua, D. australafricana, D. novem, and D. rudis. Multilocus phylogenetic analysis revealed that Chilean isolates were grouped in separate clades with their correspondent ex-types species. All species of Diaporthe were pathogenic on wounded kiwifruit after 30 days at 0°C under normal and controlled-atmosphere (2% O2 and 5% CO2) storage and they were sensitive to benomyl, pyraclostrobin, and tebuconazole fungicides. D. ambigua isolates were the most virulent based on the lesion length measured in inoculated Hayward and Jintao kiwifruit. These findings confirm D. ambigua, D. australafricana, D. novem, and D. rudis as the causal agents of kiwifruit rot during cold storage in Chile. The specie D. actinidiae, a common of Diaporthe sp. found associated with kiwifruit rot, was not identified in the present study.

Characterization of the Bull's Eye Rot of Apple in Chile.

Apple fruit in Chile are primarily produced for export to Asia, Europe, and the United States, which typically requires 15 to 40 days of maritime transportation. Therefore, Chilean apple production must fulfill the sanitization requirements imposed by the receiving countries. Under these circumstances, it was important to clarify the etiology of bull's eye rot that can severely affect 'Cripps Pink' apple and other late-harvest cultivars in Chile. Based on morphological characteristics and the partial sequence analysis of the internal transcribed spacer sequences and ß-tubulin genes, Neofabraea alba was identified as the causal agent of the bull's eye rot of Chilean apple. These results were further corroborated using species-specific primers. The incidence of bull's eye rot varied considerably; for instance, in 2009, 0.0 to 58.7% in 38 Cripps Pink orchards surveyed in the relatively arid and humid apple-growing areas of Chile, respectively. There was no evidence for the presence of N. malicorticis or N. perennans, which are commonly identified as causal agents of bull's eye rot in other apple-producing countries. Altogether, these data suggest that N. alba might represent the predominant and possibly the only cause of bull's-eye rot of Chilean apple.

Bioactive metabolites from the fungus Nectria galligena, the main apple canker agent in Chile.

The phytopathogenic fungus Nectria galligena Bres. is the most common canker disease agent of hardwood trees. The terpenoids colletochlorin B, colletorin B, ilicicolin C, E, and F, as well as the phytotoxin alpha,beta-dehydrocurvularin have been isolated from liquid cultures of N. galligena obtained from the xylem of infected apple trees in central Chile. Ilicicolin C and F and alpha,beta-dehydrocurvularin were active against Pseudomonas syringae with IC50 values of 28.5, 28.5, and 14.2 microg/mL, respectively, in the same range as streptomycin and penicillin G (11 and 15 microg/mL, respectively). All of the compounds showed moderate inhibitory activity toward the enzymes acetylcholinesterase (AChE) and beta-glucuronidase. The most active enzyme inhibitors were colletochlorin B and ilicicolin C and E, with IC50 values of 30-36 microg/mL in the AChE assay and 32-43 microg/mL in the beta-glucuronidase test. All of the chlorinated compounds showed some toxicity toward human lung fibroblasts, with IC50 values in the range of 64-120 microg/mL. alpha,beta-Dehydrocurvularin proved to be the most toxic compound, showing IC50 values less than 12 microg/mL. The effect of the isolated compounds on seed germination and radicle and epicotyl growth was assessed in lettuce and millet seeds. At 100 and 200 microg/disk, alpha,beta-dehydrocurvularin significantly reduced radicle length and epicotyl growth in Lactuca sativa. This is the first report on the occurrence of colletochlorin B, colletorin B, ilicicolin C, E, and F, as well as alpha,beta-dehydrocurvularin associated to N. galligena.